PROJECT SUMMARY A lentiviral vector (CCLc- ?AS3-FB {?AS3LV}) is being investigated for the treatment of severe sickle cell disease, however, it suffers from low titer, sub-optimal gene transfer to CD34+ hematopoietic stem cells (HSCs), and expression likely insufficient to definitively cure ?-thalassemia (although sufficient to prevent sickling in pre-clinical studies). We hypothesize that there are known and unknown human ?-globin genomic sequences within ?AS3LV that are inhibiting vector performance. Studies outlined in this proposal will investigate how removal and/or addition of known or unknown elements within ?AS3LV's human ?-globin genomic sequences affect titer, gene delivery to HSCs, and expression of the anti-sickling ?AS3-globin gene. The outcome of these studies will provide insight into how specific regulatory elements influence the performance of ?AS3LV across multiple categories. Moreover, this research will yield a second generation of improved lentiviral vectors for efficiently transferring and effectively expressing the anti-sickling ?AS3-globin gene for gene therapy of sickle cell disease.